Method of detecting procarboxypeptidase A and carboxypeptidase A levels in biological fluids

ABSTRACT

Methods of measuring carboxypeptidase A levels and total carboxypeptidase A levels, wherein procarboxypeptidase A is converted to carboxypeptidase A by addition of clostripain, in a biological fluid with a carboxypeptidase A substrate, specificity of which is enhanced by addition of a carboxypeptidase A specific inhibitor are provided. In addition, methods of diagnosing acute pancreatitis by measurement of carboxypeptidase A levels and pancreatic cancer by measurement of total carboxypeptidase A levels are also provided.

This Application is a 371 PCT/US98/06615 filed Apr. 10, 1998 and claimsbenefit of provisional Nos. 60/041,835 filed Apr. 10, 1997 and60/055,495 field Aug. 12, 1997.

BACKGROUND OF THE INVENTION

Determination of altered enzyme levels by measurement of enzyme activityin biological samples is used routinely by clinicians to assist in thediagnosis of a multitude of diseases or conditions wherein physicalsymptoms alone may not be definitive. However, the usefulness of suchassays is dependent upon the specificity of the enzyme to the disease orcondition and the sensitivity and selectivity of the enzymatic assay.

For example, acute pancreatitis is defined clinically as a discreteepisode of symptoms caused by intrapancreatic activation of digestiveenzymes. The cause of this activation is unknown; however, prematureactivation of zymogen to active enzymes within the pancreas results inautodigestion and inflammation of the pancreas. Symptoms include asteady, dull or boring pain in the epigastrium or left upper abdominalquadrant which is poorly localized and reaches peak intensity withinfifteen minutes to one hour. The incidence of acute pancreatitis isdifficult to ascertain as uniform diagnostic criteria and effort havenot been applied. However, there is an urgency in accurately diagnosingacute pancreatitis to exclude other acute conditions that requiredifferent, usually surgical, management such as perforated peptic ulcer,acute cholangitis, appendicitis and mesenteric infarction. In contrast,pancreatitis is best treated through a “hands off” approach ofeliminating food intake and increasing hydration.

Determination of serum amylase activity is the test most frequently usedfor the diagnosis of acute pancreatitis. The frequent use of this testundoubtedly stems from the ease in obtaining substrate and performingthe spectrophotometric analysis. In addition, the cost is significantlyless as compared to an ultrasound or CT scan. However, results from thisassay are difficult to interpret with any certainty due to the extensivedistribution and background levels of amylase throughout the body.Pancreatic amylase only accounts for approximately 40% of the amylasefound in serum. In fact, many individuals experience hyperamylasis forreasons unrelated to pancreatic pathology such as salivary diseases, gutdiseases, liver diseases and other conditions such as renal failure,thermal burns, alcoholism, postoperative state, ketoacidosis, fallopianor ovarian cysts, pneumonia, anorexia and abdominal aortic aneurysm.(Pieper-Bigelow et al. (1990) Gastroenterol. Clin. North Am.19:793-810).

Sensitivity of the amylase test is also suspect, in part, because of theshort half-life of the enzyme relative to others produced in thepancreas. With a half-life of only two hours, amylase is the firstenzyme to return to normal levels (Ventrucci et al. (1987) Pancreas2:506-509) resulting in a sensitivity of only 33% two days after aninitial bout of pancreatitis (Winslet et al. (1992) Gut 33:982-986).

Further, even in cases where pancreatic disease is known to be present,there is no correlation between the severity of pancreatitis and thelevel of serum amylase.

Accordingly, a number of digestive enzymes produced by the pancreas havebeen considered as possible alternatives to amylase.

Carboxypeptidase A (CPA) is a digestive enzyme synthesized exclusivelyby the pancreas as a zymogen precursor, procarboxypeptidase A (PCPA).Significant levels of CPA have been detected in serum of those sufferingfrom acute pancreatitis, while healthy individuals have little (Roth, M.and Rohner, A. (1983) Clin. Chim. Acta. 135:65-71; Kazmierczak, S. C.and Van Lente, F. (1989) Clin. Chem. 35:251-255) to no (Peterson et al.(1982) Anal. Biochem. 125:420-426; Brown et al. (1987) Anal. Biochem.161:219-225) detectable amounts of the enzyme. Several substrates and avariety of assay procedures have been proposed for the determination ofthe pancreatic enzyme CPA. Such assays include UV spectrophotometry todirectly monitor the cleavage of the peptide bond, and colorimetric andfluorometric methods to measure the amino acid released from theC-terminus. (Bergmeyer, H. U., Ed. (1974) Methods of Enzymatic Analysis,Vol. 2, 2nd ed., Academic Press, New York; Roth, M. and Rohner, A.(1983) Clin. Chim. Acta 135:65-71). More frequently used substratesinclude N-benzyloxycarbonyl-glycyl-L-phenylalanine (Z-Gly-Phe) andhippuryl-phenylalanine (Bz-Gly-L-Phe). An assay involving thedetermination of the α-naphthol released from the N-terminal blockinggroup in naphthoxycarbonyl-phenylalanine has also been disclosed.(Ravin, H. A. and Seligman, A. M. (1951) J. Biol. Chem. 190:391-402). Inaddition, a spectrophotometric assay employingN-(2-furanacryloyl)-L-Phe-L-Phe (FAPP) has been reported. (Peterson etal. (1982) Anal. Biochem. 125:420-426). However, while the FAPPsubstrate had the best kinetic constants of any CPA substrate to date,its modest change in absorbance at 330 nm (ε=2000) and the high initialabsorbance at that wavelength (ε=9350) significantly reduce thesensitivity and precision of this assay.

A new class of synthetic peptides suitable for assaying peptidaseactivity was described by Kingsbury et al. (1984) Proc. Nat'l Acad. Sci.USA 81:4573-4576. These peptides contain amino acid mimetics withnucleophilic substitutions at the α-carbon of glycine residues. Theamino acid mimetics are stable when the nitrogen lone pair electrons aredelocalized, as they are in a peptide bond, and release of the aminoacid mimetic results in its decomposition to generate the nucleophilicsubstituent. If the substituent is linked to the glycine residue throughsulfur, decomposition yields a compound with a free sulfhydryl group.Its appearance can be monitored spectrophotometrically in the presenceof Ellman's reagent which reacts rapidly and quantitatively with freesulfhydryl groups to form a highly colored anionic species that absorbsat 412 nm. (Ellman, G. L. (1959) Arch. Biochem. Biophys. 82:70-77). Anassay for measuring CPA in serum with the N-blocked phenylalaninesubstrate, N-acetyl-phenylalanyl-L-3-thiaphenylalanine was developed.(Brown et al. (1987) Analytical Biochemistry 161:219-225). However, useof an assay measuring CPA activity to diagnose acute pancreatitis hasbeen debated.

Using p-OH Bz Gly Phe as a CPA substrate, Kazmierczak and Van Lentecarried out an extensive study comparing CPA, amylase and lipase levelsas indicators of acute pancreatitis. A major difficulty for CPA wastheir finding that patients with renal insufficiency, but withoutpancreatitis, appeared to have elevated levels of the enzyme.Kazmierczak and Van Lente also found the diagnostic sensitivity of thethree assays to be comparable at cutoff values of 3 (23 μg/L), 185 and300 U/L, respectively. They concluded that automated analysis for CPAactivity, even in the absence of interferences, does not add to thediagnostic information provided by the widely available assays foramylase and lipase activity. (Kazmierczak, S. C. and Van Lente, F.(1989) Clin. Chem. 35(2):251-5). High levels of CPA were also reportedto be present in normal serum by Roth, M. and Rohner, A. (1983) Clin.Chim Acta 135:65-71. Both groups found the average value of putative CPAin normal sera to be approximately 3.9 μg/L.

Pancreatic cancer is even more difficult to diagnose than acutepancreatitis, resulting in an abysmal mortality rate since individualsfrequently seek treatment only after the disease has reached advancedstages which are accompanied by pain, weight loss and jaundice. Thecancer is rarely diagnosed in its initial stages, in part because nocost-effective, non-invasive diagnostic test exists to date. Whileultrasonography, CT scans and endoscopic retrogradecholangiopancreatography can confirm the presence of pancreatic cancer,these procedures are too expensive to use for general screening and arenormally not applied until too late. Attempts to discover a marker forpancreatic cancer have been hindered by the fact that little is knownabout risk factors which would predispose individuals to the cancer.However, a number of individuals with pancreatic cancer have beenreported to demonstrate high PCPA serum levels with normal amounts ofCPA. Accordingly, determination of elevated levels of PCPA may serve asan early screen for this disease which has the lowest survival rate ofany cancer. Like CPA, however, attempts to measure PCPA in serum haveproduced conflicting results.

Trypsin can fully activate PCPA in a concentration-dependent manner. Ithas been reported that 2 mg of trypsin per ml of serum can producemaximum activity of CPA in thirty minutes, although half as much trypsinrequired 120 hours. Amounts less than 0.5 mg per ml showed no detectableactivation. (Peterson, L. M. and Holmquist, B. (1983) Biochemistry22:3077-3082). These values are different from other studies, however,wherein maximum activity was obtained with 1 mg trypsin per ml of serum(Brown, K. S. Senior Thesis, Princeton University 1986).

Accordingly, there is a need for more sensitive and definitive enzymaticassays to diagnose diseases such as acute pancreatitis and pancreaticcancer in patients.

SUMMARY OF THE INVENTION

An object of the present invention is to provide a method of enhancingthe sensitivity and specificity of an assay measuring enzymatic activityin a sample which comprises measuring enzymatic activity in the samplein the presence and absence of a specific inhibitor of the enzymaticactivity.

Another object of the present invention is to provide a method ofmeasuring carboxypeptidase A levels in biological fluids which comprisescontacting a biological fluid with a carboxypeptidase A substrate in thepresence and absence of a carboxypeptidase A specific inhibitor andmeasuring changes in optical density resulting from hydrolysis of thecarboxypeptidase A substrate by carboxypeptidase A in the presence andabsence of the carboxypeptidase A specific inhibitor.

Another object of the present invention is to provide a method ofdiagnosing acute pancreatitis in a patient comprising detecting elevatedlevels of carboxypeptidase A in a biological fluid of a patient using acarboxypeptidase A substrate, the specificity of which is enhanced byaddition of a carboxypeptidase A specific inhibitor.

Yet another object of the present invention is to provide a method ofmeasuring total carboxypeptidase A levels, including bothcarboxypeptidase A and procarboxypeptidase A in a biological fluid whichcomprises converting any procarboxypeptidase A in the biological fluidto carboxypeptidase A by addition of clostripain; contacting thebiological fluid with a carboxypeptidase A substrate in the presence andabsence of a carboxypeptidase A specific inhibitor; and measuringchanges in optical density resulting from hydrolysis of thecarboxypeptidase A substrate by carboxypeptidase A in the presence andabsence of the carboxypeptidase A specific inhibitor.

Yet another object of the present invention is to provide a method ofdiagnosing pancreatic cancer in a patient which comprises detectingelevated levels of total carboxypeptidase A in a biological fluid of apatient by converting any procarboxypeptidase A in the biological fluidto carboxypeptidase A by addition of clostripain and contacting thebiological fluid with a carboxypeptidase A substrate, the specificity ofwhich is enhanced by addition of a carboxypeptidase A specificinhibitor, and contrasting the total carboxypeptidase level with anamount of carboxypeptidase A in the sample determined in the absence ofclostripain so that elevated levels of procarboxypeptidase A indicativeof early stage pancreatic cancer can be determined.

DETAILED DESCRIPTION OF THE INVENTION

It has now been found that the sensitivity and specificity of enzymaticassays can be enhanced by utilization of blanks containing an inhibitorspecific to the enzyme. For example, the pancreatic enzymecarboxypeptidase A was first characterized by Anson in 1937. (Anson, M.L. (1937) J. Gen. Physiol. 20:663).N-acetyl-phenylalanyl-L-3-thiaphenylalanine is a specific substrate forcarboxypeptidase A (CPA) and has been proposed as a replacement for theamylase assay in diagnosing pancreatitis. (Brown et al. (1987)Analytical Biochemistry 161:219-225). However, the extraneous opticaldensity changes associated with its application to serum determinationslimited sensitivity and reproducibility of this assay. Further, thequestion of high CPA baseline levels in the serum of healthy subjectshas been raised which is critical to diagnostic use of the assay as itdirectly reflects upon the sensitivity in which a particular assay iscapable of assessing pancreatic pathology.

It has now been demonstrated that the sensitivity and specificity of anassay measuring levels of carboxypeptidase A (CPA) in a biological fluidby addition of a CPA substrate is enhanced by utilization of a blankcontaining a CPA specific inhibitor. In a preferred embodiment of thisCPA assay, the biological fluid comprises plasma or serum. However, itis believed that this assay could also be used to determine CPA levelsin other biological fluids such as urine. Addition of CPA specificinhibitors a-benzylsuccinic acid or potato CPA inhibitor (CPI) to CPAassays with the substrate N-acetyl-phenylalanyl-L-3-thiaphenylalanine(NAcPSP) was found to correct for all extraneous changes in opticaldensity (O.D.) resulting from decomposition of the substrate orreagents. By addition of these specific inhibitors to the blank, it isnow possible to specifically measure the changes in optical densityresulting from the low amount of CPA, i.e., 0.3 μg/L, in normal humanserum. Accordingly, an assay measuring enzymatic hydrolysis of a CPAsubstrate, preferably NAcPSP in the presence and absence of a CPAspecific inhibitor, can now be used to reproducibly detect CPA levels inbiological fluids and in diagnosing acute pancreatitis.

In this assay, hydrolysis of the CPA substrate is measured usingEllman's reagent. Ellman's reagent reacts rapidly and quantitativelywith free sulfhydryl groups to form a highly colored anionic speciesthat absorbs at 412 nm. (Ellman, G. L. (1959) Arch. Biochem. Biophys.82:70-77). However, neither the substrate nor Ellman's reagent used tomeasure hydrolysis of the substrate are completely stable compounds.Accordingly, decomposition of these compounds results in an increase inO.D. during the assay. In addition, the half-Ellman which is liberatedby the interaction of the Ellman's reagent with serum albumin is subjectto reoxidation which causes a decrease in O.D. These effects are quitemodest for ordinary analytical purposes. For example, substratebreakdown is 0.63 percent per hour at 37° C. However, the O.D. increasesattributable to the minute amount of CPA in the serum of healthyindividuals is so small that these ancillary O.D. changes can bedominating. Further, a blank containing Ellman's reagent and substrateonly does not compensate for the reoxidation. Alternatively, a blankcontaining serum and Ellman's reagent does not compensate for substratebreakdown. In the assay of the present invention, however, wherein aspecific CPA inhibitor is added to the blank, all extraneous O.D.changes are corrected. In the manual assay described below, the CPAspecific inhibitor is prepared in a concentrated solution so that only avery small volume is required thus rendering any pipetting error indispensing it to have an inappreciable effect on concentrationrelationships.

Accordingly, the present invention provides a diagnostic assay forpancreatitis by measuring levels of CPA in a biological fluid such asserum using a CPA substrate, preferably NAcPSP. As will be obvious tothose of skill in the art, however, CPA substrates with a similarMichaelis constant, Vmax, and extinction coefficient to NAcPSP can alsobe used. The specificity of the substrate for the enzyme is enhanced byaddition of a CPA specific inhibitor to the blank. For example, FAPP hasbeen disclosed to produce such modest changes in absorbance that thesensitivity and precision of an assay measuring CPA in serum issignificantly reduced. However, addition of a CPA specific inhibitor tothe blank of this assay increased the sensitivity and precision suchthat FAPP is a useful substrate for determination of CPA levels in serumwith the method of the present invention. Examples of CPA specificinhibitors which can be used include, but are not limited to,α-benzylsuccinic acid (K_(i)=1 μM) and CPI. Monoclonal antibodies raisedagainst the enzyme in accordance with well known methods can also beused as specific inhibitors in the assay. By adding a specific enzymeinhibitor to the assay blank, only optical density changes attributableto enzymatic cleavage of the substrate are measured. Assays areperformed in pairs with the inclusion of a sufficient concentration ofinhibitor in the second cuvette to eliminate at least 99 percent of anyCPA activity and to correct for extraneous O.D. changes in normal serumthus making it possible to accurately detect CPA levels in serum ofhealthy adults. Assays can be performed by a manual method or in a CobasBio centrifugal analyzer (Roche Diagnostics Systems, Branchburg, N.J.)at 37° C.

Stability of CPA activity during the time period for the assay of thepresent invention was confirmed. In these experiments, sera from twoindividuals was assayed and monitored spectrophotometrically every hourover a period of six hours. Absorbance increased in a linear fashion forboth samples, thus indicating that CPA activity was in fact beingmonitored and that it was stable at 37° C. for this time period, whichis actually twice as long as the standard assay. These studies alsoindicate that, while incubation for three hours is preferred, activityafter only a one hour incubation period provides an accurate estimate ofCPA activity for diagnostic purposes since CPA activity, on average, islinear throughout the incubation period. Further, experiments in serumfrom four different individuals demonstrated a linear increase in ΔODwhich correlated with serum concentrations.

Specificity of the assay for CPA and not PCPA was also confirmed. Inthese experiments, three different samples were tested for CPA activityusing either α-benzylsuccinic acid or CPI, an inhibitor that does notbind to PCPA. Both inhibitors were added at concentrations sufficient toeliminate at least 99% of all CPA activity. In two of the samples fromhealthy individuals, CPA activity was approximately equivalent with thetwo inhibitors. The third sample, which was known to have approximately250 times more PCPA than CPA, also yielded similar results with the twoinhibitors.

Reproducibility of the assay of the present invention was determined insera from two individuals by performing numerous assays on specificserum samples. Analysis of three samples from each individual revealedthat CPA activity measurements were reproducible for both a given serumand among different samples for a given individual. Results from theseexperiments are depicted in Table 1. Enzyme activity is expressed inU/L.

TABLE 1 Trials Mean ±2S Serum #1 set 1 10 0.098 0.030 set 2 11 0.0980.037 set 3 9 0.109 0.040 Serum #2 set 1 9 0.058 0.022 set 2 9 0.0460.014 set 3 14 0.042 0.024

Variability of CPA activity measurements for a single sample is believedto be largely attributable to the reproducibility of thespectrophotometric determination in view of the exceedingly high levelsof sensitivity at which CPA is being measured. Mean CPA activity variedsomewhat among samples drawn at different times from a given individual,although all sets overlap within error demonstrating that a healthyindividual does not experience wide fluctuations in serum CPA levels.

Having established the selectivity and reproducibility of this assay, alarger study was performed on 108 samples of plasma collected fromhealthy blood donors. Using the standard assay described in Example 2with 0.1 ml serum per ml of solution, the CPA level of each sample wasdetermined. CPA activities were distributed somewhat asymmetricallyalthough they approximated a normal distribution with a mean of0.068±0.055 U/L (X±2S) and a median of 0.064 U/L.

CPA activities in males and females, analyzed independently, had similardistributions. Of the 108 samples, 62 were taken from males with anin-class mean of 0.071±0.057 U/L (X±2S) and median of 0.068, while 46females had a mean of 0.063±0.050 U/L (X±2S) and a median of 0.057 U/L.Further analysis of the data revealed no correlation between CPA levelsand age of the donors (ranging from 21 to 79 years).

The baseline of the assay of the present invention is approximately 58fold less than that reported by Kazmierczak and Van Lente as a cutoff(Kazmierczak, S. C. and Van Lente, F. (1989) Clin. Chem. 35(2):251-5).Thus, the assay of the present invention is much more sensitive than theCPA assay taught by Kazmierczak and Van Lente to probably not be“warranted” in diagnosing pancreatitis. Further, elevated levels of CPAobserved by Kazmierczak and Van Lente in patients suffering from renalinsufficiency have recently been found to be attributable to a severalfold elevation in the proenzyme without any accompanying appearance ofdetectable CPA. Therefore, contrary to teachings in the prior art, theCPA assay of the present invention is useful in diagnosing pancreatitis.

A number of patients were tested with this assay to assess levels of CPAactivity during illness. Two main groups were studied: individualsdiagnosed with pancreatitis and patients with nonpancreatic diseases.Tests on pancreatic serum were conducted with the automated assaydescribed in, Example 2. Elevations in serum CPA concentrations wereobserved in seven selected pancreatics with values as high as 1000 timesabove baseline. Tests on serum from patients with nonpancreaticconditions uncovered an enormous variety of disease states with elevatedamylase but normal CPA levels.

Accordingly, patients exhibiting symptoms of acute pancreatitis can bediagnosed quickly and easily using the assay of the present invention todetect elevated levels of CPA. Using this assay, normal CPA activitieshave been determined to have a range of 0.068±0.083 U/L (X±3SD).Accordingly, serum levels of CPA greater than 0.20 U/L are consideredelevated and are indicative of pancreatitis.

As will be obvious to those of skill in the art upon this disclosure,average CPA levels in other biological fluids from healthy controlpopulations such as plasma, saliva or urine can be routinely determinedin accordance with these teachings and elevated CPA levels in thesebiological fluids can also be indicative of pancreatitis.

Further, while the focus of these experiments was upon enhancing the CPAassay, the addition of a specific inhibitor to the blank of an enzymaticassay can be used to enhance the accuracy and sensitivity of any enzymeassay. Various specific inhibitors for selected enzymes are known in theart and can be routinely added to blanks of assays for the selectedenzymes in accordance with the teachings herein. Further, monoclonalantibodies against a selected enzyme which are also useful as specificinhibitor in the method of the present invention can be raised inaccordance with well known techniques. Accordingly, the presentinvention provides a method for enhancing the sensitivity andspecificity of any assay measuring enzymatic activity in a samplewherein enzymatic activity in the sample is measured in the presence andabsence of a specific inhibitor of the enzymatic activity.

It has also been found that the CPA assay of the present invention canbe modified to measure both CPA and PCPA, referred to herein as “totalCPA”, in a biological fluid. Several enzymes have been suggested for usein measuring levels of total CPA activity including bovine trypsin,subtilisin and urokinase, with bovine trypsin being preferred.(Peterson, L. M. and Holmquist, B. (1983) Biochemistry 22:3077-3082).However, attempts to optimize PCPA activation by trypsin by addition ofcombined reagents and soybean trypsin inhibitors after a shortincubation indicate that trypsin activated serum does not reflect actualPCPA levels due to tryptic-induced degradation of CPA.

In the present invention, the protease clostripain was added to activateany PCPA in the biological fluid so that total CPA could be measured.Tests with sera from four healthy individuals and one with pancreatitisindicated that, unlike trypsin, supramaximal clostripain concentrationsresulted in only slight CPA activity losses. Further, CPA activitiesafter clostripain activation were significantly higher than the maximumvalues that could be obtained when using trypsin.

Sixty-six samples previously tested for CPA activity were analyzed fortotal CPA activity following clostripain activation. Of these, twosamples had exceedingly high levels of PCPA and were not analyzedfurther since they fell well outside the range of “normal” based uponcalculations with the other samples. Overall, males (37) had a meanvalue for total CPA activity of 1.50±0.82 U/L (X±2S) while females (27)had a distribution of 1.50±1.04 U/L (X±2S).

Serum from patients with pancreatic cancer at various stages andpatients with related conditions were analyzed for total CPAconcentrations by the assay of the present invention wherein clostripainwas first added to convert any PCPA to CPA. A detailed description ofactivation by clostripain is provided in Example 7. Data from theseexperiments are shown in Table 2.

TABLE 2 CPA and total CPA activity in patient with pancreatic cancer andrelated diseases Total CPA CPA Patient (U/L) (U/L) Ratio Diagnosis FLC0.061 43.9 720 adenocarcinoma of head TMa 0.008 6.99 874 adenocarcinoma(lymphatics) S 0.126 5.61 45 adenocarcinoma SSa 0.090 4.97 55 islet celltumor SSb 0.038 3.27 86 adenocarcinoma VC 0.049 3.19 65 adenocarcinoma;head removed RD — 3.06 — duodenal tumor with obstructed duct TMb 0.0371.70 46 cancer of the ampulla SG 0.046 1.14 25 pancreatic tail tumorwith duct polyps GT 0.034 0.82 24 advanced, unresectable cancer DJ 0.0880.45 5 advanced adenocarcinoma

While cancer patients exhibited normal CPA levels, total CPAconcentrations were well above normal for several individuals. Thosepatients who demonstrated the highest PCPA levels generally sufferedfrom resectable, early stage cancer. In contrast, individuals withadvanced stages of cancer (GT and DJ) had very low PCPA levels, withserum from DJ containing less PCPA (0.36 U/L) than any individual in thehealthy population. Accordingly, elevated levels of PCPA determined bymeasuring total CPA using the assay of the present invention is believedto be useful as a marker in the diagnosis of early stage pancreaticcancer.

The following nonlimiting examples are provided to further illustratethe instant invention.

EXAMPLES Example 1 Materials

N-acetyl-phenylalanyl-3-thiaphenylalanine (NAcPSP) was obtained fromPeptides International (Louisville, Ky.) and stored at room temperaturein a desiccator. Bovine trypsin, clostripain, subtilisin, urokinase,Nα-benzoyl-L-arginine p-nitroanilide (BAPNA), soybean trypsin inhibitor(STI), and bovine serum albumin were all obtained from Sigma ChemicalCo. (St. Louis, Mo.) and stored over desiccant at 4° C. Ellman's reagent[5,5′-dithio-bis(2-nitrobenzoic acid)],DL-benzylsuccinic acid, andN-(3-[2-furyl]acryloyl)-phe-phe (FAPP) were also purchased from Sigmaand stored at room temperature. Carboxypeptidase Potato Inhibitor (CPI)and dithiothreitol (Cleland's Reagent) were obtained from Calbiochem(San Diego, Calif.) and stored over a desiccant at 4° C. All otherchemicals were of analytical grade.

Solutions of NAcPSP, FAPP, Ellman's reagent, α-benzylsuccinic acid,clostripain and bovine CPA were stored at −20° C. All other solutionswere made on the day of the experiment and kept at 0° C.

Blood and plasma used to assess baseline CPA and PCPA levels wascollected in three sets from blood donors at New York Hospital. Otherblood samples were obtained from either the NYU Hospital or the McCoshClinic at Princeton University. All samples were stored at −20° C.

Pancreatic juice was obtained from the externalized pancreatic duct inan otherwise healthy individual and stored at −20° C.

Example 2

Standard Assay for Measuring CPA activity in Human Serum

CPA activity was determined by applying the substrate NAcPSP to asolution containing Ellman's reagent and serum and subsequentlymonitoring the production of the half Ellman's anion at 412 nmspectrophotometrically. This assay employed the following reactants atthe indicated final concentrations: human serum (usually 0.1 ml per mltotal solution); Ellman's reagent (0.5 mM); Tris buffer, pH 7.5, (0.2M); NaCl (0.5 M); and NAcPSP (0.4 mM). Serum concentrations ranging from0.003 to 0.15 ml of total solution were also applied. The total volumeof the reactants was three ml.

The addition of Ellman's reagent to the serum was followed by a fiveminute incubation at 22° C., which allowed free sulfhydryl groups in theserum to react to completion with Ellman's reagent. After the additionof NAcPSP, one ml of this mixture was dispensed into each of two plasticcuvettes with one-cm path lengths. One of these cuvettes (referred toherein as the blank) contained one microliter of a CPA inhibitor (0.4 Min the case of benzylsuccinate) and the other cuvette (referred toherein as the test) contained a microliter of water. The cuvettes werethen sealed and incubated at 37° C. Absorbance was monitored every houror hour and a half for a total of three hours with a Zeiss model PM6spectrophotometer (Thornwood, N.Y.).

This determination of CPA levels was based upon the difference inabsorbance between the blank and the test cuvettes for a given sampleand presented as units of activity per liter of serum in accordance withthe following calculation. A unit is defined as one micromole ofhalf-Ellman's anion produced per minute. Since Ellman's anion has amolar extinction coefficient of 13,600 L/Mol-cm at 412 nm, theproduction of one μmole per ml of solution results in an increase inabsorbance of 13.6. $\begin{matrix}{{1\quad {U/L}} \approx \quad {{\left( {1\quad {{\mu mole}/\min}} \right)/L}\quad {serum}}} \\{\approx \quad {{\left( {13.6\quad \Delta \quad {{OD}/\min}} \right)/L}\quad {serum}}} \\{\approx \quad {{\left( {0.00136\quad \Delta \quad {{OD}/\min}} \right)/0.1}\quad {ml}\quad {serum}}}\end{matrix}$

After 3 hours, the change in absorbance is 0.245 relative to a blankwith no CPA activity for 1 U/L with the standard assay.

An automated assay using the Cobas Bio analyzer was also performedwherein a first set of determinations was made without the inhibitor. Asecond set was then made wherein the substrate solution contained theinhibitor. In the automated assay, the analyzer was set to provide for afive minute reaction time of the Ellman Reagent with the serum afterwhich substrate was added and optical density determinations were madeevery 10 seconds for five minutes at a wavelength of 412 nm.

Example 3

Determination of Kinetic Constant of CPA substrates

Kinetic constants of serum CPA were determined for NAcPSP by utilizingthe standard procedure and modifying the concentration of substratewithin the range of 0.1 to 0.8 mM. Experiments were also performed usingbovine CPA, pancreatic juice, and pseudocyst fluid, with each applied inconcentrations high enough so that initial velocity calculations couldbe made following change in absorbance for two minutes. All CPA sourceswere additionally tested with FAPP in order to obtain values for Km andrelative velocities. Assays of FAPP hydrolysis involved the addition ofenzymes to solutions containing FAPP (0.02 to 0.2 mM) in a buffer of 50mM Tris (pH 7.5) and 0.45 M NaCl. The reaction was followed at 330 nmusing a Zeiss model PM6 spectrophotometer. At this wavelength thesubstrate has a high initial absorbance (ε=9350) which declines as thereaction proceeds (Δε=2000). (Peterson et al. (1982) Anal. Biochem.125:420-426).

Lineweaver-Burke analysis was used to evaluate Michaelis constants forCPA with the two substrates. Independent confirmation of these constantsfor FAPP were made using competitive inhibition analysis. NAcPSPhydrolysis was allowed to proceed following introduction of CPA for oneminute, at which point a small, concentrated volume of FAPP was added tothe solution. The change in velocity which resulted was used tocalculate a K_(i) of FAPP against NAcPSP.

Example 4

Comparison of CPA Inhibitors

Solutions of a-benzylsuccinic acid with a reported K_(i) of 1 μM and 1.6μM (Byers, L. D. and Wolfenden, R. (1973) Biochemistry 12:2070-2078;Peterson et al. (1976) Biochemistry 15:2501-2508), and CarboxypeptidasePotato Inhibitor (CPI), with a reported K_(i) of 5 nM (Hass, G. M. andRyan, C. A. (1980) Biochem. Biophys. Res. Commun. 97:1481-1486) wereprepared such that each solution had similar efficiency at eliminatingCPA activity as determined by their ability to arrest the activity of 50ng bovine CPA assayed using the standard procedure described in Example2. Final concentrations of the 1 μl addition of the two solutions were0.4 M for α-benzylsuccinic acid and 1.4 mM for CPI. To compare theireffectiveness in eliminating serum CPA activity, an identical amount ofeach inhibitor solution was added to two sets of three different serumsamples, which were assayed by the standard procedure.

Further tests were also performed with α-benzylsuccinic acid atdifferent concentrations. In these tests, a higher volume of inhibitorsolution applied to the blank cuvette was always paralleled with anidentical increase in water added to the test cuvette.

Example 5

Bovine Trypsin Activity in Normal and Serum Titrated Solutions

Activities of bovine trypsin solutions were assessed by monitoring thehydrolysis of N-benzoyl-Arg-p-nitroanilide (BAPNA) at 406 nm with aZeiss model PM6 spectrophotometer. BAPNA was used at a finalconcentration of 0.2 mM in 10 mM Tris buffer (pH 8.0). Following theaddition of specified amounts of trypsin to the substrate solution,absorbance was followed over a period of two minutes with readings takenevery 15 seconds. The procedure was used to determine activity ofpancreatic juice, human serum, human serum spiked with bovine trypsin,and clostripain.

Slight modifications of this protocol were made when testing for trypsinactivity in the presence of serum. NaCl, Tris buffer (pH 8.0), and BAPNAat a final concentration of 0.5 M, 6 mM, and 0.2 mM, respectively, wereadded to a plastic cuvette containing 0.025 ml of serum for a totalvolume of 1 ml. A concentrated trypsin solution (5 mg trypsin/ml 10 mMTris buffer, pH 8.0) was added to the mixture in amounts ranging from0.4 to 4 mg trypsin per ml serum. Absorbance was monitoredspectrophotometrically at 406 nm every fifteen seconds for threeminutes.

Example 6

Activation of PCPA in human serum with trypsin

Activation of PCPA with bovine trypsin was performed in accordance withmethods described by Peterson, L. M. and Holmquist, B. (1983)Biochemistry 22:3077-3082. Bovine trypsin was dissolved in 10 mM Trisbuffer (pH 8.0) and added to provide a final concentration of 0.5 to 6mg/ml serum. Following a thirty minute incubation at 37° C., total CPAactivity was determined using the standard assay described in Example 2.Similar experiments were performed wherein PCPA was activated withsubtilisin and urokinase.

Alternatively, trypsin was added after all other reagents had been addedto the serum to provide substrate enhanced protection for the CPA. Morespecifically, trypsin was added after the addition of NAcPSP to theserum containing the Ellman's reagent. The mixture was then incubatedfor five minutes at 37° C. at which time soybean trypsin inhibitor (STI)was added in sufficient quantity (approximately 2 mg STI/5 mg trypsin)to eliminate all bovine trypsin activity. Following this step, thesolution was separated into two cuvettes as described in Example 2 andmonitored at 412 nm for 3 hours with a Zeiss model PM6spectrophotometer.

Example 7

Activation of PCPA in Human Serum with Clostripain

Approximately 3 mg (500 units) of lyophilized clostripain was dissolvedin 1 ml of 10 mM Tris buffer (pH 8.0) along with CaCl₂ and Cleland'sReagent at final concentrations of 20 mM and 1 mM, respectively. Thismixture was incubated at room temperature for approximately 2 hours.This solution was then added to serum in concentrations ranging fromapproximately 50 to 500 units of clostripain/ml of serum. One unit wasdefined as the amount of clostripain which could cleave one micromole ofNα-benzoyl arginine ethyl ester (BAEE) in one minute. Determination ofPCPA levels in blood donor samples was performed with 250 units ofclostripain per ml serum. The standard serum volume was 0.013 ml serumper ml total solution. This mixture was incubated for five minutes at37° C., after which time Ellman's Reagent was added at the standardconcentration to eliminate clostripain as it quickly derivatizes allsulfhydryl groups in the mixture. From this point, the standard assay asdescribed in Example 2 was followed.

What is claimed is:
 1. A method of measuring total carboxypeptidase Alevels in a biological fluid comprising: (a) converting anyprocarboxypeptidase A in a biological fluid to carboxypeptidase A byaddition of clostripain; (b) contacting the biological fluid with acarboxypeptidase A substrate in the presence and absence of acarboxypeptidase A specific inhibitor; and (c) measuring changes inoptical density resulting from hydrolysis of the carboxypeptidase Asubstrate by carboxypeptidase A in the biological fluid in the presenceand absence of the carboxypeptidase A specific inhibitor.
 2. A method ofdiagnosing early stage pancreatic cancer in a patient comprising: (a)converting any procarboxypeptidase A in a biological fluid obtained froma patient to carboxypeptidase A by addition of clostripain; (b)measuring total carboxypeptidase A levels in the biological fluid bydetecting changes in optical density resulting from hydrolysis of acarboxypeptidase A substrate by any carboxypeptidase A in the biologicalfluid in the presence and absence of a carboxypeptidase A specificinhibitor; and (c) determining whether the measured levels of totalcarboxypeptidase A in the biological fluid of the patient are increasedas compared to total carboxypeptidase A levels in a healthy populationdue to elevated procarboxypeptidase A in the biological fluid.